The polymerase chain reaction (PCR) is a method that is finding ever increasing use in the molecular biology and fields related thereto. The method of PCR was first described in the mid-1980s. See Mullis et al., Cold Spring Harbor Symp. Quant. Biol. (1986) 51:263, Saiki et al., Science (1985) 230:1350; and Mullis & Faloona, Methods Enzymol. (1987) 155:335. PCR finds use in a variety of applications, including the generation of probes for DNA of both known and unknown nucleic acid sequences, the generation of cDNA libraries, DNA sequencing, analysis of mutations, chromosome crawling, and the like.
Generally, PCR results in the amplification of a segment of DNA that lies between two regions of known sequence. In PCR, two oligonucleotides are used as primers for a series of synthetic reactions that are enzymatically catalyzed by a DNA polymerase. The primers are complementary to regions on opposite strands of the DNA and flank the region of DNA to be amplified. The template DNA is first denatured by heating the DNA in the presence of an excess of primer and the dNTPs. The mixture is then cooled to provide for primer annealing and extension. The cycle of denaturation, annealing and synthesis is repeated a plurality of times, which results in the production of an amplified amount of template DNA.
One limitation of PCR methods is a lack of fidelity, i.e. misincorporation of base pairs during the synthesis step. While for certain applications mispairing of bases may not pose a serious problem, for other applications low fidelity renders PCR practically useless. While methods have been developed which provide for improved fidelity, such improvements typically are concomitant with a decrease in efficiency.
Thus, there is a continued need for the development of improved methods of PCR, where the development of PCR methods which provide both high fidelity and efficiency is of particular interest.
Relevant Literature
The polymerase chain reaction is reviewed in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) Chapter 14. Other references reviewing PCR include: Ling et al., PCR Methods and Applications (1991) 63-69; Eckert & Kunkel, Nuc. Acids Res. (1990) 18:3739 and Eckert & Kunkel, PCR Methods and Applications (1991) 63-39.
Braithewaite & Ito, Nuc. Acids Res. (1993) 21:787-802 provide a review of known DNA polymerases.
Barnes, Proc. Natl. Acad. Sci. USA (1994) 91:2216-2220 reports the results of using a combination of Klentaq and Pfu polymerases in PCR amplification.